Y. D. Park et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4106–4110
4109
A
D
B
C
E
F
25 µm
50 µm
50 µm
Figure 4. Histological staining of the mouse brain sections. Staining with 9n (B and C) and staining with thioflavin T (E and F) in APP/SP1 transgenic AD model mice. Staining
with 9n (A) and thioflavin T (D) in control normal mice.
aggregate (Kd = 1.84
l
M). In principle, a good fluorescent probe for
BAY 94–9172 and AV-45, and both these molecules possess rigid
structures of stilbene (BAY 94–9172) and styrylpyridine (AV-45).
We planned to develop fluorescent markers for detecting Ab aggre-
gates by displacing the phenyl group in stilbene with pyridazin-
3(2H)-one to create styrylpyridazin-3(2H)-ones. Furthermore, all
of the PET imaging probes in clinical trials (BAY 94–9172, AV-45,
and GE067) contain a monomethylamino or dimethylamino group
that confers binding specificity to Ab aggregates.
Ab plaques should have high fluorescence responsiveness and a
strong binding affinity for aggregated Ab peptide.16 Upon binding
to Ab plaques, the fluorescent probe should undergo a significant
change in its fluorescent properties because an increase in fluores-
cence intensity means that the probe will be ‘turned on’ upon
interacting with its target. The probe 9n meets these requirements,
detecting Ab aggregates with an FAb/F0 = 40.84 and a Kd = 1.84 lM.
Encouraged by these promising properties, we further tested
the applicability of compound 9n for the fluorescent imaging of
intracellular aggregated Ab in SHSY5Y neuroblastoma cells. To
determine the intracellular uptake and aggregation of Ab, Ab42
In conclusion, we show that dimethylamino group-containing
the 2-styrylpyridazin-3(2H)-one derivative (9n) has fluorescence
characteristics appropriate for imaging Ab aggregates. Compound
9n exhibited a strong fluorescence response (FAb/F0 = 40.84) and
(1
l
M) labeled with fluorescein isothiocyanate (FITC-Ab42) was
binding affinity (Kd = 1.84 lM) to Ab aggregates, and it clearly
incubated with SHSY5Y cells for 3 days and then imaged with fluo-
rescence microscopy. The uptake of FITC-Ab42 was observed in
SHSY5Y cells (Fig. 3B). Previous reports have suggested that extra-
cellular soluble Ab is taken up and then forms high-molecular-
weight aggregates of Ab42 (>200 kDa).17 To determine whether
compound 9n could be used as the basis of a fluorescence probe
for detecting intercellular Ab aggregates, cells loaded with unla-
beled Ab42 were sonicated with SDS, and homogenates were incu-
stained both intracellular Ab aggregates and Ab plaques in the
transgenic AD model mice (APP/PS1). Thus, 2-styrylpyridazin-
3(2H)-one derivatives may be good candidates as alternative fluo-
rescence imaging agents for the study of AD. Based on the above
results, our next study will focus on radiolabeled 2-styrylpyrida-
zin-3(2H)-one derivatives as potentially useful PET imaging agents
for cerebral Ab plaques.
This work was supported by a National Research Foundation
(NRF) grant funded by the Korean government Ministry of Educa-
tion, Science and Technology (MEST).
bated with 10 lM of 9n for 10 min. Fluorescent precipitates were
visible by microscopy in the Ab-loaded cell extracts (Fig. 3D). The
labeling pattern was consistent with that observed with thioflavin
T (Fig. 3F). Extracts from cells grown in the absence of Ab failed to
show fluorescent staining (Fig. 3C and E). These staining experi-
ments strongly suggest that 2-styrylpyridazin-3(2H)-one-based
probes are capable of staining intercellular Ab aggregates.
To assess whether the 2-styrylpyridazin-3(2H)-one-based
probe 9n could stain amyloid deposits in AD mouse brain tissue,
we tested 9n in brain sections from 15-month-old transgenic AD
model mice (APP/PS1). Figure 4 shows representative fluorescence
micrographs of these tissue samples. Ab plaques were clearly
stained with 9n, consistent with its high affinity for Ab aggregates
in in vitro assay (Fig. 4B and C). The labeling pattern was consistent
with the signal obtained from standard thioflavin T-stained sec-
tions (Fig. 4E and F). In contrast, tissue from normal mice did not
stain with 9n or thioflavin T (Fig. 4A and D). These results suggest
that 9n can function as a probe for detecting Ab plaques in the
brain.
References and notes
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Previously, stilbene derivatives were identified as ligands with
high binding affinities to Ab aggregates.18 Currently, two structur-
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Park, Yong Dae
